1. Field of the Invention
This invention relates to a method for diagnosing for abnormalities in the biosynthesis of type III procollagen in cultured skin fibroblasts. More particularly, this invention relates to a method for diagnosing for aneurysms which comprises incubating cultured fibroblasts with labeled proline to form labeled procollagen, subjecting the labeled procollagen to pepsin digestion, and determining the ratio of type I collagen to type III collagen in the mixture.
2. Description of the Background
The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference. For convenience, the disclosures are referenced in the following text and respectively grouped in the appended bibliography.
Aneurysms are sacs formed by the dilatation of the wall of an artery, a vein, or the heart. Based on post mortem examinations, 4.5% to 6.6% of the population has an abdominal aortic aneurysm (Fievez, 1989). The underlying causes of aneurysm development are not known, although atherosclerosis, hypertension, and smoking have been suggested as possible factors which either cause or aggravate the development of the disease. In addition, a clear genetic predisposition to the development of aneurysms has been demonstrated in some patients (Johansen and Koepsell, 1986; Tilson add Seashore, 1984 a; b; Cole et al., 1989; Bengtsson et al., 1989, Darling et al., 1989).
Aneurysmal dilatations are commonly seem among the elderly. However, a variety of mutations in the primary structure of type III procollagen can lead to aneurysms in young adulthood (Superta-Furga et al., 1988; Tromp et al., 1989; Kontusaari et al., 1990; and Kontusaari et al., 1990), as demonstrated by the genetic mutations responsible for the rare disorder Ehlers Danlos syndrome IV (EDS IV). Many of the genetic mutations seen in EDS IV patients are associated with alterations in the steady state levels, thermal stability, or secretion of the type III procollagen synthesized by cultured skin fibroblasts (Superta-Furga et al., 1989; Stolle et al., 1985).
The pathogenesis of arterial aneurysms is likely to be multifactoral. Structural alterations in lamellar architecture (Zatina et al., 1984), altered protease levels (Busuttil et al., 1980; Busittil et al., 1982), copper deficiency (Tilson, 1982), elastin abnormalities (Keeley et al., 1989), and hemodynamic factors (Zarins and Glagov, 1982) have all been implicated in the etiology of aneurysms.
In addition, familial clustering of aneurysms (Johansen and Koepsell, 1986, Tilson and Seashore 1984 a; b) suggests that in some patients a heritable factor is involved. Approximately 19% of abdominal aortic aneurysm patients have a documented family history of the disease (Johansen and Koepsell, 1986). However, the number of individuals with a genetic predisposition to the disease may be much greater (Pope et al., 1983; Tilson, 1990).
Mutations in collagen genes may play a role in diseases such as aneurysms (Prockop and Kivirikko, 1984; Prockop, 1985) and several investigators have reported a reduction of type III collagen in skin and pepsin digested media from cultured skin fibroblasts of some patients with cerebral aneurysms (Poe et al., 1981; Neil-Dwyer et al., 1983; de Paepe et al., 1988). A decreased type III collagen content of the aortic wall of patients with a family history of abdominal aortic aneurysms has also been reported (Menashi et al., 1987). Other reports, however, have shown no significant alterations in either the synthesis or quantity of type III collagen associated with non EDS IV related aneurysms. For example, normal biosynthesis of type III procollagen has been demonstrated by cultured skin fibroblasts from a proband in a family with three members having cerebral aneurysms (Leblanc et al., 1989). Similarly, the type III collagen content of the aortic walls of abdominal aortic aneurysm patients, either with or without a family history of the disease, were found to be comparable to age matched controls (Rizzo et al,, 1989).
Proteolytic enzymes have been reported to be useful as probes for the triple-helical conformation of procollagen. These enzymes digest all nonhelical regions in triple-helical collagen molecules as well as non-collagenous molecules (Bruckner et al., 1981).
Patients with the bone disorder osteogenesis imperfecta have been found to secrete a type I procollagen which lacked pro-alpha2(I) chains and consisted of a trimer of pro-alpha1(I) chains (Deak et al., 1983). The pro-alpha2(I) chains were postulated to have a mutated structure in the carboxyl-terminal propeptides reducing their affinity to form trimers with the pro-alpha2(I) chains. The trimer of pro-alphal(I) chains was found to have decreased thermal stability presumably due to the absence of pro-alpha2(I) chains (Deak et al., 1985).
In a study on 10 patients with the connective tissue disorder Ehlers-Danlos type IV syndrome (EDS IV), 9 patients were found to secrete a decreased amount of type III procollagen and one patient was found to secrete a structurally altered type III procollagen (Stolle et al., 1985).
DNA tests for mutations in the type III procollagen were reported to be helpful to identify individuals at risk for familial aortic aneurysms (Kontusaari et al., 1990).